The prohormone convertases PC1/3 and PC2, encoded by the genes PCSK1 and PCSK2 respectively, are the endoproteolytic enzymes responsible for the liberation of opioid-active peptides from larger precursor proteins. Prohormone convertases play important roles not only in opioid peptide-mediated pain signaling but also function in many other neuronal circuits, including in reward pathways and in hypothalamic circuits involved in feeding and energy homeostasis. For example, both rare and common variations in PCSK1 function as major risk factors for human obesity, potentially due to deficiencies in hypothalamic peptidergic processing. In collaboration with clinicians who have identified children with novel mutations in PCSK1, we have recently determined that mutant human and mouse PC1/3 proteins are subject to targeting defects which are likely to result in hypothalamic proteostatic stress. Based on our prior finding that mouse PC1/3 proteins oligomerize during synthesis, we propose that dominant-negative interactions play a major role in human PC1/3 heterozygote obesity phenotypes, affecting precursor processing to bioactive peptides involved in satiety signaling. We propose that external stressors will exacerbate even mild forms of PC1/3 conformational distress, impairing C-terminal cleavage of PC1/3 to the smaller, more active forms. These processes will ultimately converge to strongly impair precursor processing, eg. proopiomelanocortin cleavage to beta-endorphin, ACTH, and most importantly, to the anorexic peptide ?- MSH. In the present proposal we will use CRISPR-engineered cell models to elucidate the cell biology and precursor processing efficacy of three human PC1/3 variants and mutants known to be strongly associated with increased risk of obesity. Secondly, we will create mouse models of two common human PCSK1 obesity mutants, and a third model of a rare but highly impaired mutant, to extend findings made in cell culture to actual secretory tissues, and to identify the specific physiologic alterations which underlie the PCSK1-mediated obesity phenotype. Lastly, we will test our hypothesis that processing deficits in proopiomelanocortin-synthesizing neurons underlie the obesity phenotype by selectively eliminating Pcsk1 expression in proopiomelanocortin-expressing cells using a floxed Pcsk1 null mouse model. The results of these studies will illuminate the biosynthetic mechanisms controlling hypothalamic peptide production that contribute to human susceptibility to a variety of diseases, from obesity to reward pathways in drug addiction.